Monoclonal Antibodies and Single Chain Antibody Fragments Against Cell-Surface Prostate Specific Membrane Antigen

ABSTRACT

Isolated monoclonal antibodies or an antigen binding portion thereof which bind to prostate specific membrane antigen in its native form occurring on the surface of tumor cells characterized in that it is linked to a label or a cytotoxic agent or constructed as a part of a bispecific antibody or a recombinant diabody.

Cancer of the prostate is the most commonly diagnosed cancer in men andthe second most common cause of death in the Western civilization. Nocurative treatment currently exists for this tumor after progressionbeyond resectable boundaries. Because of the significant mortality andmorbidity associated with disease progression, there is an urgent needfor new targeted treatments. In contrast to cancer in other organsystems, prostate cancer represents an excellent target for antibodytherapy for a number of reasons, that include i) the prostate expressestissue specific antigens, ii) the prostate is a non-essential organ andits destruction will not harm the host, iii) the sites of metastasis arelymph nodes and bone that receive high levels of circulating antibodies,and iv) the PSA serum levels provide a means to monitor therapeuticresponse.

Among several candidate markers that have been identified for prostatecancer, prostate specific membrane antigen (PSMA) seems to be mostprominent. This type II transmembrane glycoprotein of about 100 KDconsists of a short intracellular segment (amino acids 1-18), atransmembrane domain (amino acids 19-43), and an extensive extracellulardomain (amino acids 44-750). PSMA may serve as a useful target forimmunotherapy because it meets the following criteria: i) expression isprimarily restricted to the prostate, ii) PSMA is abundantly expressedas protein at all stages of disease, iii) it is presented at the cellsurface but not shed into the circulation, iv) expression is associatedwith enzymatic or signaling activity. PSMA is also expressed in theneovasculature of most other solid tumors, and therefore may be a targetfor specific anti-angiogenetic drug delivery.

Because of their target-oriented specificities, a lot of emphasis hasbeen put on the development of monoclonal antibodies (mAbs) fordiagnostic and therapeutic applications in cancer medicine. However, thein vivo use of mAbs is associated with serious problems, because oftheir size and immunogenicity. Therefore, research has focused on thedevelopment of smaller therapeutic antibodies with fewer side effects,better tumor accessibility and faster clearance rates. Geneticengineering has made it possible to construct single chain antibodyfragments (scFv) which are potentially powerful tools for cancertherapy. These small antibodies are composed of the variable domains ofthe light chain (V_(L)) and the heavy chain (V_(H)) connected by alinker peptide. They show little immunogenicity, almost no toxiceffects, an increased clearance rate, an improved uptake by the tumorand a better penetration into the tumor cells. Recombinant murine scFvcan be established according to standard methods using either expressionlibraries from hybridomas or spleen cells of specifically immunized mice[Chowdhury et al., Mol. Immunol. 4 (1997), p. 9-20].

The first published mAb (7E11-C5) against PSMA targets at theintracellular domain of the protein and was shown to be highly prostatespecific [Horoszewicz et al., Anticancer Res. 7 (1987), p, 927-935].Also, monoclonal antibodies against the extracellular domain of PSMAhave been raised after immunization with the antigen. However, there isstill a discrepancy between binding to the antigen on fixed cells andhistological sections on the one hand and binding to viable tumor cellson the other hand.

Prostate specific membrane antigen (PSMA) is a prostate marker that ishighly expressed in normal prostate as well as in prostate cancer. Itsexpression is increased in prostate cancer and is found primarily in theprostate.

Prostate specific membrane antigen (PSMA) is a unique membrane boundcell protein which is over expressed manifold on prostate cancer as wellas in the neovasculature of many other solid tumors, but not in thevasculature of the normal tissues. This unique expression of PSMA makesit an important marker as well as a large extracellular target ofimaging agents. PSMA can serve as target for delivery of therapeuticagents such as cytotoxins or radionuclides. PSMA has two uniqueenzymatic functions, folate hydrolase and NAALADase and it is found tobe recycled like other membrane bound receptors through clathrin coatedpits.

A radio-immuno-conjugate form of the anti-PSMA monoclonal antibody (mAb)7E11, is commercially available as “ProstaScint®” which is currentlybeing used to diagnose prostate cancer metastasis and recurrence. ThePSMA epitope recognized by monoclonal antibody 7E11-C5.3 is located inthe cytoplasmic domain of the prostate specific membrane antigen.

There are, however, also reports describing PSMA expression innon-prostatic tissues including kidney, liver and brain. A possibleexplanation therefore is provided by O'Keefe et al., (Prostate, 2004,February 1; 58 (2) 200-10), namely that there is a PSMA-like gene whichpossesses 98% identity to the PSMA gene at the nucleotide level, whichis expressed in kidney and liver under the control of a differentpromoter to the PSMA gene.

WO 01/009192 describes the development of human monoclonal antibodies toprostate-specific membrane antigen. Human anti-PSMA monoclonalantibodies were generated by immunizing mice with purified PSMA orenriched preparations of PSMA antigen. Such purified antigen is adenatured PSMA since it has been purified by immunoadsorption after celllysis with ionic detergents.

WO 97/35616 describes monoclonal antibodies specific for theextracellular domain of prostate-specific membrane antigen. Theimmunizations were performed with a C-terminal peptide or aPSMA-expressing tumor membrane preparation. The mAbs do not bindspecifically to PSMA-expressing cells and can therefore not be used fordiagnostic or therapeutic purposes.

Bander et al., Seminars in Oncology, 2003, pp 667-677 and US2004/0213791 respectively disclose monoclonal antibodies directedagainst prostate-specific membrane antigen. Since the immunization wasperformed with purified antigen, the monoclonal antibodies do not have ahigh cell binding and no scFv could be obtained from neither of thesemAb.

WO 98/03873 describes the same antibodies as in US 2004/0213791 orbinding portions thereof which recognize an extracellular domain ofprostate-specific membrane antigen. It could not be shown that thebinding portions of the antibodies do in fact bind to the antigen.

Fracasso et al., The Prostate, 2002, pp 9-23 describe anti-PSMAmonoclonal antibodies which are chemically coupled to thericine-A-chain. The constructs described in this article do not bindsufficiently specific to the target and have the generally describeddisadvantages of generation on immunotoxins.

It is one object of the present invention to provide superior means andconstructs which help to differentiate with higher reliability betweentumor cells and healthy cells which do express PSMA or a similar proteinand PSMA-negative cells. Such constructs can be used to target morespecifically prostate cancer.

It is another object to provide constructs which destroy specificprostate cancer cells which express PSMA.

Prostate-specific membrane antigen (PSMA) is an attractive target forimmunotherapy of prostate cancer. However, on prostate cells PSMA isexpressed with a specific tertiary and quaternary structure andantibodies elicited with isolated denatured PSMA do not efficientlyrecognize PSMA expressing tumor cells. Antibodies and scFv binding todenatured PSMA can be obtained after immunization with the isolatedpurified antigen. The present invention, however, allows the generationof high affinity antibodies and scFv against native cellular PSMA by adifferent immunization method which gives only a poor yield of positiveclones. Only the later antibodies elicited with native PSMA may reactwith cell-surface PSMA and can be used as diagnostic and therapeutictools.

Monoclonal antibodies (mAbs), single chain antibody fragments (scFv) anddiabodies of the present invention were prepared according toconventional methods from mice spleen cells. However, the mice had beenimmunized with LNCaP cells and LNCaP cell lysate containing full-lengthnative PSMA. In a preferred embodiment of the present invention theantigen, namely the full length native PSMA has been obtained aftertreatment of the cells, preferably LNCaP cells with a special lysisbuffer called M-PER, mammalian protein extraction reagent which iscommercially available from Pierce, Roquefort, Ill. The M-PER bufferuses a proprietary detergent in 25 mM bicine buffer (pH 7.6). Hybridomasand scFv were screened and selected by flow cytometry on PSMA-positiveLNCaP cells after absorption with PSMA-negative DU 145 prostate cells.Additionally, they were tested for reactivity with purified PSMA.Resulting monoclonal antibodies and scFv were characterized by flowcytometry on LNCaP and PSMA-transfected DU 145 and by western blot withpurified glycosylated and deglycosylated PSMA. In addition,immunocytology with LNCaP cells and immunohistochemistry on paraffinsections of prostate cancer samples was prepared.

In the course of the present invention three mAbs (3/F11, 3/A12 and3/E7) could be obtained, that were reactive with viable LNCaP cells andPSMA-transfected DU 145 cells but not with other cell lines notexpressing PSMA. Binding to LNCaP cells was very strong. At saturationconcentrations (100 nM) the mean PE fluorescence intensity (MFI) wasbetween 1000 and 1600. Reactivity with purified PSMA was stronger withthe native form (ELISA) than with the denatured and deglycosylatedprotein (western blot). Immunohistochemistry on paraffin sections wasspecifically positive for epithelial cells with mAb E7.

From the mAb 3/A12 two scFv, called E8 and A5, were obtained byselection of recombinant phages on LNCaP cells and purified PSMA. Thesequence of scFv E8 was identical to a scFv A4, which was obtained fromthe B-cell library of the same mouse. ScFv E8 was strongly reactive withLNCaP cells showing a MFI of about 100 at saturation concentrations,whereas the MFI of scFv A5 was only about 40 under the same conditions.No or minimal binding was seen with other cell lines lacking PSMAexpression. Binding of both scFv to purified denatured glycosylated anddeglycosylated PSMA was weak. Furthermore, from mAb 3/F11 the scFvcalled D7 and for mAb 3/E7 the scFv called H12 was obtained.

In the present application we describe three mAb, which are differentfrom those published by other authors with respect to high bindingaffinity and high staining of PSMA expressing prostate cancer cells. Theantibodies 3/F11, 3/A12 and 3/E7 do not only show a strong bindingactivity but also internalization into LNCaP cells as shown byimmunofluorescence cytology and detection with confocal laser scanningmicroscopy. These mAbs were obtained after immunisation with full lengthnative PSMA, which is in contrast to different published immunisationmethods.

After immunization with purified denatured PSMA mAbs were obtained whichwere highly specific for the antigen, but had only a limited binding toPSMA expressing LNCaP cells and also were not internalized into thecells. These control data are not shown in the present application.There are a few anti-PSMA mAbs described in literature. However, themean fluorescence intensity values were much lower than with theantibodies of the present invention.

Similarly to the mAbs, anti-PSMA scFv were generated after immunisationwith denatured and native PSMA. With the denatured PSMA we obtained scFvhighly specific to the antigen, but not binding to LNCaP cells (data notshown in the present application). In contrast, with native PSMA weobtained scFv with a high cell binding activity, but a poor binding tothe isolated denatured antigen.

However, the problems identified in this and other trials withchemically linked immunotoxins are the development of antibodies againstthe immunotoxins, liver toxicity and vascular leak syndrome and alsodifficulties in producing large quantities of defined material. Theseproblems are, at least in part, overcome by using recombinant DNAtechnology which makes the construction of less immunogenic and smallerimmunotoxins feasible, and more easily permits the production ofimmunotoxins in large quantities. It is also believed that penetrationinto tumors should be better for small proteins than large conjugates.Therefore, recombinant immunotoxins were engineered by fusing the codingsequence of the scFv E8, H12, D7 and A5 and the toxin PE40. The centralfinding was that all recombinant immunotoxins effectively killedcultured prostate cancer cells in a dose dependent manner. Strongkilling was found not only with the highly binding E8—with IC50 of about0.05 nM, but also with the lower binding A5-fusion protein with IC50 ofabout 0.09 nM. Killing of not PSMA expressing prostate cancer cells wasmore than 2000-fold less. This may be traced back to residual bacterialproteins or other toxic agents in the immunotoxin preparations, becausethe same background could be observed in equally high concentrationswith the scFv alone. The term IC50 is defined as the concentration in nMof the toxin which reduces cells proliferation to 50% of the cellproliferation without adding a toxin.

The antibodies and scFv described in this application specifically bindto native cell-surface PSMA and therefore will have value in diagnosticand therapeutic applications focusing on PSMA as a target antigen forprostate cancer.

Since PSMA is expressed on prostate cancer cells with a specifictertiary and quaternary structure, only antibodies against this cellularconformation may recognize and strongly bind to viable prostate cancercells and PSMA-expressing tissue. Therefore, the aim of the presentstudy was to generate such mAbs and scFv that can be used fortherapeutic and diagnostic targeting of prostate cancer.

The present invention provides therefore an isolated monoclonal antibodyor an antigen binding portion thereof which binds to prostate specificmembrane antigen in its native form occurring on the surface of tumorcells which is linked to a label or a cytotoxic agent.

The term “isolated monoclonal antibody” refers to a glycoproteincomprising at least two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated as V_(H)) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains, namely CH1, CH2 and CH3. Each light chain contains a lightchain variable region (V_(L)) and a light chain constant region (C_(L)).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, which are also called complementarity determiningregions (CDR) interspersed with regions that are more conserved. Thoseregions are also called framework regions (FR). Each V_(H) and V_(L)region is composed of three CDRs and four FRs arranged from aminoterminus to carboxy terminus in the following order: FR1, CDR1, FR2,CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chainscontain a binding domain that interacts with an antigen.

In FIGS. 13, 14 and 20, 21 the CDRs are marked by grey boxes. Thoseareas are important for the binding of the monoclonal antibody or theantigen binding portion thereof. The other areas are framework regionswhich can be replaced by other sequences, provided the three-dimensionalstructure which is required for binding is not disturbed. In case thestructure of the construct is changed, there will be no sufficientbinding to the antigen. Monoclonal antibodies derived from mouse maycause unwanted immunological side-effects due to the fact that theycontain a protein from another species which may elicit antibodies. Inorder to overcome this problem the monoclonal antibodies or the antigenbinding portions thereof may be humanized. The process of humanizingmonoclonal antibodies is known to the person skilled in the art. Theframework regions of a mouse mAb are replaced by the corresponding humanframework regions. In order to maintain the preferred binding propertiesthe sequences of the CDRs should be maintained as far as possible. Itmay be required, however, to perform some amino acid changes in order tooptimise the binding properties. This can be performed by the personskilled in the art by standard proceedings. Furthermore by introducing ahuman framework it may be necessary to perform amino acid changes and/ordeletions in order to improve the properties of the construct.

The term “antigen binding portion” of the monoclonal antibody refers toone or more fragments of such an antibody which retained the ability tospecifically binding to the prostate specific membrane antigen in itsnative form. Examples of antigen binding portions of the antibodyinclude a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and C_(H1) domains, an F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region, an Fd fragment consisting of the V_(H) and C_(H1)domain, an F_(V) fragment consisting of the V_(L) and V_(H) domains of asingle arm of an antibody, a dAb fragment which consists of a V_(H)domain and an isolated complementarity determining region (CDR).

The isolated monoclonal antibody or antigen binding portion thereofaccording to the present invention can preferably be internalized by atumor cell if it is used for therapeutic purposes. For diagnosticpurposes an internalisation may not be required.

The isolated monoclonal antibody or an antigen binding portion thereofaccording to the present invention binds strongly to LNCAP cells but notto cells which lack expression of prostate specific membrane antigen.

The binding of the isolated monoclonal antibody or antigen bindingportion thereof is measured by PE fluorescence intensity (MFI) which ispreferably equal or higher than 40 for an scFv and preferably higherthan 1000 for an mAb at saturating concentrations.

The binding properties of the isolated monoclonal antibodies or anantigen binding portion thereof to the native PSMA were compared bytreating LNCAP cells with increasing concentrations of the first stepanti-PSMA Ab followed by incubation with the second step PE-labeledantibody. From the resulting saturation curves the antibodyconcentration reaching 50% saturation of PSMA sites can be read. Thethree mAb 3/F11, 3/A12 and 3/E7 showed a high binding activity reaching50% saturation of PSMA sites at approximately 16 nM (3/F11), 2 nM(3/A12) and 30 nM (3/E7). With the scFv a 50% saturation of PSMA siteswas found at 10 nM (E8) and 60 nM (A5).

In order to determine the binding strength the PE (phycoerythin)fluorescence intensity (MFI) was measured. The MFI values were plottedagainst the antibody (or binding fragments thereof) concentrationwhereby the plateau value of MFI corresponds to 100% saturation withantigen. After having determined the top value (plateau corresponding to100% saturation of antigen) the value corresponding to 50% of saturationcan be easily determined. By using the graph the correspondingconcentration of the antibodies or binding fragments thereof in nM canbe seen.

The isolated monoclonal antibody or an antigen binding portion thereofcomprises a label which may be a particle which emits radioactiveradiation. This particle may be a radioactive element in a form whichcan be linked to the construct, preferably in the form of a complex. Forexample an mAb labeled with ¹¹¹Indium may be used as aradioimmunoscintigraphy agent in the detection of distant metastatictumors in prostate cancer patients. Of course other suitable radioactiveelements like ³⁵S or ¹³¹I can be used.

Alternatively the isolated monoclonal antibody or antigen bindingportion thereof may comprise a cytotoxic agent which is a cell toxicsubstance selected from the group consisting of toxins, for exampletaxol, cytocalasin B, gramicidin D, ethidium bromid, emetine, mitomycin,etopside, tenopside, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy antracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosteron, glycocorticoids, procain,tetracaine, lidokaine, propranolol and/or puromycin.

In a preferred embodiment of the present invention an isolatedmonoclonal antibody or an antigen binding portion thereof comprises apartial amino acid sequence of at least 10 consecutive amino acids ofSEQ ID NO:1 (scFv E8), SEQ ID NO:10 (scFv A5), SEQ ID NO:20 (scFv H12)and/or SEQ ID NO:22 (scFv D7). In a preferred embodiment the monoclonalantibody or antigen binding protein thereof comprises at least 25 or,more preferred, at least 35 and most preferred at least 50 consecutiveamino acids of SEQ ID NO:1, SEQ ID NO:10, SEQ ID NO:20 and/or SEQ IDNO:22, respectively.

In a preferred embodiment the isolated monoclonal antibody or antigenbinding portion thereof comprises at least one of the CDRs having SEQ IDNO:2-SEQ ID NO:7 and/or SEQ ID NO:11 to 16. More preferably suchconstruct comprises at least 3 and more preferably at least 5 of thoseCDR sequences. In a similar manner the CDRs can be deduced from FIGS. 20and 21 wherein the CDR sequences are designated.

It is a further aspect of the invention to provide DNA sequences whichcan be used for the preparation of monoclonal antibodies or bindingfragments thereof. SEQ ID NO:8 and 9 relate to scFv E8 and SEQ ID NO:17and 18 relate to scFv A5. SEQ ID NO:19 and 23 relate to scFv H12 and SEQID NO:21 and 24 relate to scFv D7. The sequences report the codingstrand and the complementary strand thereto. SEQ ID NOS:9 and 18 areshown in the 5′→3′ orientation. The polynucleotides of the presentinvention comprise a contiguous sequence of at least 20, preferably 50and more preferably 75 and most preferred at least 100 nucleotides ofthe group consisting of SEQ ID NOS: 8, 9, 17, 18, 19, 21, 23 and 24. Thesequence coding for the CDR are in particular preferred.

It is one aspect of the present invention to provide a pharmaceuticalcomposition comprising an isolated monoclonal antibody or an antigenbinding portion thereof as described in the present application. Thepharmaceutical composition of the present invention comprises themonoclonal antibody or an antigen binding portion thereof together withpharmaceutically acceptable additives. Preferably such a composition isprepared for intramuscular or intraveneous injection. Alternatively theantibody may be provided in a depot formulation which allows thesustained release of the biologically active agent over a certain periodof time which may range preferably from one to six months. Such asustained release formulation may comprise a biodegradable polymer likea polylactide or polylactide/polyglycolide copolymer which is degradedover a prolonged period of time in the human body whereby the antibodyor the antigen binding portion thereof preferably having a toxine isreleased in a controlled manner over a certain period of time.

The isolated monoclonal antibody or an antigen binding portion thereofmay be used for the preparation of a medicament for the treatment ofcancer, in particular prostate cancer.

Alternatively the invention provides a diagnostic kit for the detectionof tumor cells comprising an isolated monoclonal antibody or an antigenbinding portion thereof. In such embodiments the label allows thedetection of the construct with suitable detection devices.

The invention provides also a method for the in vitro identification oftumor cells by which the tumor cells to be identified are contacted withan isolated monoclonal antibody or an antigen binding portion thereofwhich carries a label which can be detected by suitable analyticaldevices. The label allows the diagnostic identification of tumor cells,for example in section of human tissues obtained after surgery orbiopsy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: FACS-analysis of the mAb 3/F11, 3/A12 and 3/E7 binding to thesurface of PSMA-expressing LNCaP cells at saturation concentrations

FIG. 1 a-c: Antigen saturation curves of mAb 3/F11 (a), 3/A12 (b), 3/E7(c)

FIG. 2: Immunfluorescence cytology: Binding of a) mAb 3/F11 b) mAb 3A/12c) 3E7 to LNCaP cells. The left pictures show a control staining with4′,6-Diamidino-2-phenylindole (DAPI).

FIG. 3: Immunfluorescence cytology: Internalization of a) mAb 3/F11 b)mAb 3A/12 c) 3E7 in LNCaP cells. The left pictures show control stainingwith 4′,6-Diamidino-2-phenylindole (DAPI).

FIG. 4: Western blot with purified PSMA and the mAbs 3/E7, and 3/A12 and3/F11

FIG. 5: Western blot with glycosylated and deglycosylated PSMA and mAb3/A12

FIG. 6: Immunohistochemistry of mAb 3/E7 on a paraffin section ofprostate cancer

FIG. 7 a/b: FACS-analysis of the scFv E8 (a), and A5 (b) onPSMA-expressing LNCaP cells at saturation concentrations

FIG. 7 c/d: Antigen saturation curves of scFv E8 (c) and A5 (d)

FIG. 8: Western blot with purified PSMA and the scFv A5 and E8

FIG. 9: immunocytology of scFv E8 on LNCaP cells

FIG. 10: construct of the immunotoxin E8-P40

FIG. 11: Cytotoxic effect of recombinant immunotoxin E8-P40 on LNCaPcells FIG. 12: Cytotoxic affect of recombinant fusion protein A5-P40 onLNCaP cells

FIG. 13: Sequence of scFv E8. DNA sequence is given as well as aminoacid sequence whereby the region of the CDWs is identified by a markedarea.

FIG. 14: Sequence of scFv A5. DNA sequence is given as well as aminoacid sequence whereby the region of the CDWs is identified by a markedarea.

FIG. 15: This Figure shows binding of the scFv A5, H12 and D7 toPSMA-negative DU145 cells (A) and PSMA-positive LNCaP cells (A5=B,H12=C, D7=D). Cells were stained with the mAbs and a PE-conjugatedanti-mouse IgG mAb. Histograms represent logarithms of PE fluorescenceon flow cytometer. Negative control was done with secondary antibodyonly.

FIG. 16: The binding of the scFv A5, H12 and D7 to PSMA-negative BOSCcells (A) and PSMA-transfected BOSC cells (A5=B, H12=C, D7=D). Cellswere stained with the scFv anti-c-myc mAb and PE-conjugated anti-mouseIg. Histograms represent logarithms of PE fluorescence on flowcytometer. Negative control was done with secondary antibody only.

FIG. 17: demonstrates the cytotoxic effect of recombinant immunotoxineHE12-PE40 on LNCaP (black) and DU cells (white).

FIG. 18: shows schematically the construction scheme of the A5-CD3diabody.

FIG. 19: shows the cytotoxic effect of a diabody constructed from scFvA5 (A5/CD3) at different concentrations and peripheral blood lymphocytes(effect or target ratio 10:1) on LNCaP cells after 48 h incubation.

FIG. 20: shows the sequence of scFv H12. The amino acid sequence isgiven in the one-letter-code in the first line (corresponding to SEQ IDNO:20). The coding strand is shown on the second line (SEQ ID NO:19) andthe complementary strand is shown in the third line. This sequencecorresponds to SEQ ID NO:23. The CDRs are specifically designated as CDRH1, H2, H3, L1, L2 and L3. The nucleic acid sequences coding for the CDRregions are shown on a grey background.

FIG. 21: shows the sequence of scFv D7. The amino acid sequence is shownon the first line in the one letter code. This sequence corresponds toSEQ ID NO:22. The coding nucleic acid strand is shown on the first line.This sequence corresponds to SEQ ID NO:21 and the complementary strandis shown on the third line. This sequence corresponds to SEQ ID NO:24.The CDR regions H1, H2, H3, L1 and L2 are shown in the sequence. Thenucleic acid sequences coding for those regions are shown on a greybackground.

The present invention is further illustrated by the following examples.

EXAMPLE 1 a) Preparation of PSMA

The human prostate carcinoma cell lines LNCaP, DU 145, PC-3 and HeLa aswell as the hybridoma 7E11-C5.3 (IgG1-k, PSMA) were purchased from theAmerican Type Culture Collection (ATCC), Rockville, Md., USA. LNCaP, DU145 and HeLa were cultured in RPMI 1640 medium, PC-3 in F12 Nutrimixmedium, both supplemented with penicillin (100 000 U/l), streptomycin(100 mg/l) and 10% FCS at 37° C. in a humidified atmosphere of 5% CO₂.For the generation of LNCaP cells expressing unglycosylated PSMA ontheir surface 2 μg/ml tunicamycin (ICN Biomedicals) were added to themedium for 48 h.

Fixed LNCaP cells were obtained by treatment with 4% paraformaldehydefor 10 min at RT, and then thoroughly washing with PBS.

For preparing purified PSMA, 10⁸ LNCaP cells were washed with PBS andthen lysed in PBS containing 1% IGEPAL for 20 min at room temperature.After centrifugation at 10,000 g the supernatant was given on a 7E11-C5affinity chromatography column (Amersham Biosciences, Uppsala, Sweden)and PSMA was eluted with 100 mM glycine buffer pH 2.5 containing 1%Triton X-100. After neutralisation the protein was extensively dialyzedwith PBS.

For preparation of deglycosylated PSMA, 1/10 vol glycoprotein-denaturingbuffer (BioLabs), was added to the solution with purified PSMA andheated for 10 min at 100° C. Then 1/10 vol 10% NP-40 (10%) and 50 UPNGase per μg PSMA was added and incubated at 37° C. for 1 h.

For preparation of a LNCaP cell lysate containing full length nativePSMA, cells were lysed with M-PER reagent (Pierce) for 10 min and thencentrifuged at 15,000 rpm for 30 min at 4° C. The supernatant containingnative full length PSMA was collected (M-PER-lysate). The high molecularfraction (100 to 600 KD) of this lysate was separated by HPLC on aBiosil 250 size exclusion column.

b) Transfection of Full Length PSMA into DU 145 and PC3 Cells

Full length PSMA was cloned in two fragments (fragment 1 from bp 262 tothe unique EcoRI restriction site at bp 1573 and fragment 2 fromposition 1574 to 2512) into the vector pCR3.1 (Invitrogen). Transienttransfection was obtained by adding a mixture of 4 μg DNA and 10 μlLipofectamine (Invitrogen) in 500 μl RPMI medium to 10⁶ cells accordingto the manufacturer's protocol. After 48 h incubation the transienttransfected cells were used for testing.

EXAMPLE 2 Immunization of Mice

Four-month old female Balb/c mice were immunized intraperitoneally with300 μg M-PER lysate from LNCaP cells or with the high molecular HPLCfraction of the lysate, or with 106 LNCaP cells, fixed with 2%paraformaldehyde. These preparations were mixed 1:1 with completeFreund's adjuvant. Each mouse received 4 or 5 immunizations at 2-weekintervals. Four days after the last immunization spleen cells werecollected and either used for the preparation of hybridomas or a B-celllibrary.

EXAMPLE 3 Preparation of a B-Cell Library

The mouse spleen was washed in phosphate buffered saline (PBS), mincedto small pieces, washed again in PBS and then gently homogenized in a“loose-fitting” hand homogenizer. The resulting single cell suspensionwas overlayered onto Ficoll (Pharmacia, Freiburg, Germany) andcentrifuged at 400 g for 20 min at room temperature. Interphase B cellswere isolated with CD19 microbeads according to the manufacturer'sinstructions (Miltenyl, Bergisch Gladbach, Germany). 10⁶ B-cells werelysed in 350 μl of a solution consisting of 4 M guanidine thiocyanate,25 mM sodium citrate, 0.5% sodium N-laurosylsarcosinate and 100 mM2-mercaptoethanol.

EXAMPLE 4 a) Preparation of Hybridomas

The spleen was aseptically removed and a single cell suspension wasprepared in RPMI-1640 medium without serum. The splenocytes were addedto SP2/0 myeloma cells at a ratio of 10:1 and the fusion and selectionwas performed to established procedures [Galfre et al., Nature (1979),p. 131-133].

Hybridoma supernatants were tested by FACS on LNCaP and DU145 cells andby an ELISA with purified PSMA as solid phase. Monoclonal antibodieswere purified using a protein G column (Pharmacia).

b) Isotype Determination of the mAbs

Ig-isotypes of the anti-PSMA mAbs were determined by ELISA using eitherunlabelled (solid phase) or peroxidase-labeled (tracer) anti-isotypespecific antibodies (Southern Biotechnology Associates, Birmingham,Ala.).

c) Isolation and Characterization of Anti-PSMA Conformational MonoclonalAntibodies

From Balb/c mice which were immunized 5 times with the M-PER-lysate fromLNCaP cells, spleen cells were fused with SP2/0 cells according toestablished methods. Positive hybridomas were selected by flow cytometrywith LNCaP cells and ELISA on purified PSMA. By this way three positiveclones were obtained. The corresponding mAbs with their isotypes were3/F11 (IgG2a), 3/A12 (IgG1) and 3/E7 (IgG2b).

d) Characterization of mAbs

By flow cytometry it could be observed that the three mAbs and stainedLNCaP cells bind very well with a percentage of positive cells rangingfrom 95% to 98%. The shape of the curves of fluorescence versus numberof events suggested that PSMA is homogeneously distributed within theLNCaP cell population (FIG. 1). To evaluate the binding specificity ofthe anti-PSMA mAbs, PSMA-negative DU145, PC3 cells, HeLa and Jurkatcells were also stained and analyzed by flow cytometry. All three mAbsdid not stain the PSMA-negative cells (percentage of positive cellsranging from 0.04% to 2%).

The binding properties of the three antibodies were compared by treatingLNCaP cells with increasing concentrations of the first step anti-PSMAmAb followed by incubation with a saturating amount of a second stepPE-(phycoerythin)-labeled goat antibody followed by cytofluorometryanalysis. At antigen saturation concentrations [100 nM] the correctedmean PE (phycoerythin) fluorescence intensity was about 1000 for mab3A12, and about 1400 for mAb 3F11 and about 1600 for mAB 3E7. As shownfor mAb 3A12 the MFI was 5-fold lower on LNCaP cells expressingunglycosylated PSMA (grown with tunicamycine).

By immunofluorescence cytology and detection with a laser scanningconfocal microscope a strong binding of the three mAbs to LNCaP cells(FIG. 2) and also an internalization into these cells could be shown(FIG. 3). All mAbs were positive in an ELISA with purified PSMA as solidphase. With denatured PSMA the mAbs showed a signal at about 100 KD inwestern blot (FIG. 4) whereas the blot with deglycosylated PSMA was weakgiving a signal at about 84 KD, which corresponds to literature data(FIG. 5).

Immunohistochemistry on paraffin sections of prostate cancer waspositive with mAb 3/E7 but not with mAbs 3/F11 and 3/A12 (FIG. 6). Dataare summarized in Table 1.

TABLE 1 Characterization of 3 monoclonal antibodies against cell-surfacePSMA FACS FACS PSMA- Blot LNCaP transf. DU* ELISA Blot degl.Immunohisto- Hybridoma Isotype [MFI]* [MFI] PSMA PSMA PSMA chemistry3/F11 IgG2a 1400 105 pos pos (pos) neg 3/A12 IgG1 1000 110 pos pos (pos)neg 3/E7 IgG2b 1600 90 pos pos (pos) pos MFI = mean fluorescenceintensity at scFv concentration reaching antigen saturation (backgroundstaining with secondary antibody alone is subtracted) (pos) = slightlypositive

From these data it is concluded that the 3 mAbs show a very strong andhighly specific binding to native and denatured PSMA. Although thebinding to deglycosylated PSMA is weaker, a sugar specificity can beexcluded from the fact that no binding is seen to cells that do notexpress PSMA.

EXAMPLE 5 Preparation of a scFv Expression Library in the Phagemid pSEX

From the B-cell library or from hybridoma cells total RNA and mRNA wasisolated with silicagel-based membranes (Rneasy, Qiagen, Hilden,Germany) according to the manufacturer's protocol. cDNA synthesis wasperformed at 42° C. for 60 min in a final volume of 50 μl whichcontained 25 μl of denatured RNA, 10 μl 5× buffer (Promega, Heidelberg,Germany), 5 μl of 10 mM dNTP (dATP, dCTP, dGTP, dTTP, Promega), 1.5 μlRNAsin (40 U/μl, Promega) 2.5 μl of 150 pM random hexamer primers, and2.5 μl of AMV reverse transcriptase (10 U/μl, Promega). Then theencoding regions of the heavy-chains and the gamma and kappa chains wereamplified by PCR as previously described by Orum et al. [Nucleic AcidsRes. (1993), 4491-4498]. For each chain 25 separate reactions werecarried out by combining 25 different constant region forward primerswith one corresponding reverse primer. The amplified products for thelight chains and the heavy chains were purified by agarose gelelectrophoresis.

The PCR products for the light chains were digested with MluI and NotI,and ligated into the phagemid pSEX81 [Dübel et al., Gene (1993), 97-101]using a molar ratio of 1:3 (2 μg vector, 400 ng insert). The products ofone ligation were used for the electroporation of 50 μl electrocompetentE coli XL1 blue cells (Stratagene) according to the supplier's protocol.The bacteria were plated on nine 80 mm diameter agarose platescontaining 100 μg/ml ampicillin and 0.1 M glucose (SOB-AG) of andincubated overnight at 30° C. Bacteria were isolated by adding 3 ml 2xYTmedium on each plate, scraping them off with a sterile glass spreaderand pelleted at 3,000 g for 15 min. From these bacteria plasmid DNA wasisolated which revealed the VI sublibrary. Then the PCR products for theheavy chain and the VI sublibrary were digested with NcoI and HindIII.Ligation was prepared at a ratio of 3:1 (2 μg sublibrary and 400 nginsert). Transformation by electroporation, plating and collection oftransformed bacteria was done as described for the VI sublibrary. Fromnine 80 mm diameter SOB-AG plates a total of 18 ml V_(H)V_(L) librarywas obtained.

EXAMPLE 6 Production and Selection of Antibody-Displaying Phage a)Production

In the V_(H)V_(L) library in phagemid pSEX the antibody genes are fusedin frame with gene III, which encodes the minor surface protein gulp ofthe filamentous phage. Therefore, production of recombinant phagemidparticles displaying the antibody on the surface requires infection ofthe phagemid-carrying bacterial cell with the replication defectivephage M13KO7 [14]. M13KO7 was added to a 10 ml library culture at amultiplicity of 10. After incubation at 37° C. for 90 min the cells werepelleted and resuspended in 15 ml 2xYT-medium containing 100 μg/mlampicillin: 10 μg/ml tetracycline and 50 μg/ml kanamycin. The culturewas incubated overnight at 37° C. at 250 rpm, then chilled on ice andcentrifuged to remove cells. The supernatant containing the phages wasmixed with ⅕ volume of an aqueous solution containing 20% PEG 8,000 and14% NaCl and incubated 1 h at 4° C. Then a centrifugation step of 30 minat 4° C. and 6,200 g added. The pellet containing the phages wasresuspended in 2 ml 10 mM Tris/HCl pH 7.5, 20 mM NaCl, 2 mM EDTA pH 7.5and used for panning.

b) Panning to Select for Antigen- and Cell-Binding Clones

Panning on purified PSMA was done in 96 well Maxi-Sorb microtiter plates(Nunc) which were coated with a solution of purified PSMA (100 μl/well,12 μg/ml PSMA in PBS) and blocked with 4% non-fat milk/PBS. One ml ofpurified recombinant phages (circa 10¹¹) were incubated in 1 ml 4%non-fat milk/PBS supplemented with 15 μl 10% Triton X100 for 15 min andthen allowed to bind to 8 wells coated with PSMA for 2 h at 37° C. After20 rounds of washing with PBS/Tween (0.1%) the bound phages were elutedwith 0.1 M Glycin-Puffer pH 2.2. For panning on viable LNCaP cellsphages were previously absorbed on DU 145 cells. For this procedure 1 ml(circa 10¹¹) recombinant phages were incubated in 2% non-fat milk/PBSfor 15 min and then with 10⁷ DU 145 cells for 1 h at room temperature ona shaker. Then the cells were centrifuged and the supernatant with nonabsorbed phages was incubated with 106 LNCaP cells for 1 h at roomtemperature on a shaker. After 10 washing rounds with 2% non-fatmilk/PBS and 5 rounds with PBS the bound phages were eluted with 50 mMHCl with subsequent neutralization with 1 M Tris-HCl (pH 7.5). E. coliTG1 cells were infected with the eluted phages, plated on SOB-AG platesand incubated overnight at 30° C. An aliquot of the eluate was used fortitration. The selection procedure was repeated three to six times.

c) Small Scale Phage Rescue

From the titration plate 96 individual colonies were isolated and eachtransferred into one well of a 96-deep-well microtiter plate filled with500 μl 2xYT medium containing 100 μg/ml ampicillin and 0.1 M glucose(YT-AG) and incubated overnight at 37° C. (master plate). Then 40 μl ofsaturated culture from each well of the master plate were transferred tothe corresponding well of a new plate containing 400 μl of 2×YT-AGmedium.

To each well about 1×10¹⁰ M13KO7 helper phages were added and incubatedon a shaker for 2 hours at 37° C. Then the plate was centrifuged and thepellet suspended in 2xYT medium supplemented with 100 μg/ml ampicillin,10 μg/ml tetracycline, and 50 μg/ml kanamycin and incubated at 29° C.and 240 rpm overnight. After centrifugation the supernatant containingthe rescued phagemids was removed and used for phage ELISA and flowcytometry.

d) Phage-ELISA

Microtiter plates were coated with purified PSMA (1.5 μg PSMA/ml PBS)overnight and then blocked with 2% non-fat milk/PBS. To each well 200 μlof rescued phagemids, preincubated 1:1 with 2% non fat-milk/PBS, wereadded and incubated for 2 h at room temperature. After five washingsteps with PBS-Tween, bound phages were detected with 200 μl/wellanti-M13 antibody conjugated to horseradish peroxidase (Pharmacia) for 2h at room temperature. Development was carried out with3,3′,5′,5′-tetramethylbenzidine as substrate.

e) Isolation and Characterization of Anti-PSMA Conformational scFv

For generation of anti-PSMA conformational scFv a V_(H)V_(L) library inthe phagemid pSEX was constructed from the B cell library of a mouseimmunized with M-PER-lysate of LNCaP cells. This library had acomplexity of 10⁷. In a similar way a V_(H)V_(L) library was preparedfrom the monoclonal antibody 3/A12, which was obtained from the samemouse immunized with LNCaP lysate. This V_(H)V_(L) library had acomplexity of 10⁶. To isolate phages displaying cellular PSMA bindingscFv on their surface, six rounds of panning were performedalternatively on LNCaP cells after absorption with DU-145 cells inpolystyrene tubes and in microtiter plates coated with 20 μg/ml purifiedPSMA. After three, four and six panning rounds isolated phagemidcolonies were grown and phage particles were rescued by infection withM13KO7. Analysis of 800 phage clones from the B-cell library by flowcytometry with LNCaP cells and ELISA on purified PSMA showed onepositive clone called E8. Out of the V_(H)V_(L) library from mAb 3/A12two positive clones were obtained after the fourth panning round calledA4 and A5. By sequencing it was found that A4 was identical to E8.

The coding region of the scFv E8 and A5 were transferred from thephagemid pSEX into the expression vector pHOG, containing C-terminalc-myc and His-tags. The sequences with the corresponding CDRs are givenin FIG. 13 and FIG. 14. The regions coding for the CDR's of the antigenbinding portions are marked in FIGS. 13 and 14. Those sequences shouldnot be changed whereas the other parts of the sequence which are notmarked can be changed. The appropriate three-dimensional structure must,however, be maintained.

The scFv E8 strongly reacted with viable LNCaP cells as measured by flowcytometry with MFI values of about 100 at saturating concentrations,whereas binding of A5 was much weaker with MFI-values of about 40 atsaturating concentrations (FIG. 7). In contrast, binding to purifiedPSMA as solid phase in an ELISA was weak for E8 and somewhat strongerfor A5. A similar pattern was seen in western blots with denaturedglycosylated and deglycosylated PSMA (FIG. 8). By immunofluorescencecytology with LNCaP cells and detection by confocal laser microscopy avery good binding of the scFv E8 and internalization could be shown(FIG. 9). Data of the scFv are summarized in Table 2.

TABLE 2 Characterization of 2 scFv against cell-surface PSMA FACS FACSPSMA- Blot LNCaP transf. DU ELISA Blot degl. ScFv Origin [MFI] [MFI]PSMA PSMA PSMA E8 = A4 B-cell 100 70 pos (pos) (pos) library and mAbA12A5 MAbA12 40 pos pos (pos) MFI = mean fluorescence intensity at scFvconcentration reaching antigen saturation (background staining withsecondary antibody alone is subtracted) (pos) = slightly positive

EXAMPLE 7 ScFv Expression and Purification

ScFv fragments were expressed in E coli XL1-Blue (Stratagene) using thesecretion vector pHOG 21 which contains the sequences for the His-6 andc-myc-tag in a C-terminal position of the scFv [Kipriganov et al., J.Immunol. Methods (1997), p. 69-77]. E. coli bacteria transformed withpHOG plasmids were grown overnight in 2×YT-AG-medium, then diluted 1:20and grown as 600 ml cultures at 37° C. When cultures reached OD 0.8,bacteria were pelleted by centrifugation at 1,500 g for 10 min andresuspended in the same volume of fresh YT medium containing 50 :g/mlampicillin, 0.4 M sucrose and 1 mM IPTG. Then growth was continued atroom temperature for 18 to 20 h. Cells were harvested by centrifugationat 5,000 g for 10 min and 4° C. To isolate soluble periplasmic proteins,the pelleted bacteria were resuspended in 5% of the initial volume ofice-cold 50 mM Tris-HCl, 20% sucrose, 1 mM EDTA pH 8.0. After a 1 hincubation on ice, the spheroblasts were centrifuged at 20,000 g at 4°C. for 30 min yielding soluble periplasmic extract in the supernatant.The periplasmic extract was concentrated using Amicon YM10 membraneswith a 10 kDa cut-off (Amicon, Witten, Germany) followed by thoroughdialysis against 50 mM Tris-HCl, 1 M NaCl, pH 7.0.

Purification was achieved by immobilized metal affinity chromatography.This was performed using a 1 ml column of chelating Sepharose(Pharmacia) charged with Cu²⁺ and equilibrated with a buffer containing50 mM Tris-HCl and 1 M NaCl, pH 7.0. The periplasmatic extract wasloaded, washed with twenty column volumes of equilibration buffercontaining 30 mM imidazole and then eluted with the same buffercontaining 250 mM imidazole. Eluted material was dialyzed against PBS.

Determination of the protein content was performed with the Micro BCAProtein Reagent Kit (Pierce) according to the manufacturer'sinstructions.

Protein induction was obtained with IPTG and the scFv yield from a 600ml E. coli XL1 culture was about 20 μg.

EXAMPLE 8 Flow Cytometry

LNCaP, DU 145, and PC3 cells were freshly harvested from tissue cultureflasks and a single cell suspension was prepared in PBS with 3% FCS and0.1% NaN₃. Approximately 10⁵ cells were incubated with 50 μl of rescuedphagemids, preincubated 1:1 with 2% non-fat milk/PBS, 1 h on ice. After3 rounds of washing with PBS 25 μl/well anti-c-myc monoclonal antibody9E10 (10 μg/ml; Becton Dickinson) or when phages were tested 25 μl/wellanti-M13 antibody (10 μg/ml; Pharmacia) were added and incubated 40 minon ice. After washing 3 times with PBS the cells were incubated with 100μl of PE-labeled goat anti-mouse IgG (Becton Dickinson) for 40 min onice. The cells were then washed again and resuspended in 100 μl of asolution containing 1 μg/ml propidium iodide (Sigma, Deisenhofen) in PBSwith 3% FCS and 0.1% NaN₃ in order to exclude dead cells. The relativefluorescence of stained cells was measured using a FACScan flowcytometer and the CellQuest software (Becton Dickinson Mountain View,Calif.).

EXAMPLE 9 Immunofluorescence Cytology

LNCaP cells were grown on glass coverslips for 24 hours. For fixation,cells were treated with 2% paraformaldehyde in PBS for 30 min at RT,which does not permeabilize the cell membrane, washed with 1% BSA-PBS,quenched for 10 min in 50 mM NH₄Cl in PBS, and rinsed with 1% BSA-PBS.Primary monoclonal antibody at 4 μg/ml in 1% BSA-PBS was added andincubated for 60 min at 4° C. FITC-labeled goat anti-mouse secondaryantibody (2 μg/ml; Southern Biotechnology Associates Inc. Birmingham,USA) was incubated for 30 min and washed extensively with 1% BSA-PBS.Slides were mounted in Vectashield (Vector Laboratories, Inc.Burlingame, Calif.).

For internalization experiments the primary antibody was incubated for30 min at 37° C. before fixation of the cells with 2% paraformaldehydeand permeabilization with 0.5% Triton X100 in PBS.

EXAMPLE 10 a) Immunohistochemistry

Paraffin tissue sections were first deparaffinized and then treated with0.3% Triton X100 in PBS for antigen retrieval. Kryostat sections werefixed in cold acetone. The sections were treated 30 min at with 3% H₂O₂and 10% methanol for quenching of endogenous peroxidase. After blockingwith 1% BSA-PBS the primary antibody was added at a concentration of 2μg/ml and incubated for 1 h at RT. For the scFv a secondarymouse-anti-c-myc antibody was added for 1 h at RT. Then a biotinylatedgoat-anti-mouse antibody was incubated for 30 min at RT and finallydeveloped with ABC-reagent (Vectastain).

b) Western Blot Analysis

Western blot analysis was performed following sodium dodecylsulfate-polyacrylamide (SDS) gel electrophoresis of purified PSMA andcell lysate from LNCaP cells and transferred to polyvinylidenedifluoride membranes. The blots were blocked overnight in PBS containing5% non-fat milk and incubated with the purified mAbs or scFv atconcentrations of 10 μg/ml for 1 h. Then the blots were washed 5 timeswith PBS-Tween (0.5%) and incubated with horseradish peroxidaseconjugated goat anti-mouse IgG for 1 hour at RT. After 5 washes withPBS-Tween (0.5%) the blots were developed by using3,3′,5′,5′-tetramethylbenzidine as substrate.

EXAMPLE 11 Construction, Expression and Purification of scFv-PE40Proteins

The toxin used in our approach was the truncated version of Pseudomonasexotoxin (PE40), lacking domain Ia and containing only domains Ib, II,and III [Pastan et al., J. Biol. Chem. (1989), p. 15157-15160]. The DNAwith the coding region in the vector pSW200 was obtained from Prof. W.Wels, Frankfurt [Wels et al., Biotechnology (1992), p. 1128-1132]. TheDNA fragment from bp position 253 to 613 encoding PE40 was amplified byPCR from plasmid pSW200. The amplified DNA was then ligated into thevector pHOG-His-scFv in a C-terminal position to the scFv using therestriction site XbaI. All cloning steps were performed according tostandard methods in E. coli XL1 blue and the products were confirmed bysequencing.

Protein induction of the immunotoxin and purification by IMAC was thesame like the scFv. The products were tested and characterized bySDS-page, western blot and flow cytometry.

EXAMPLE 12 Cytotoxicity of scFv-PE40 Immunotoxins

The metabolism of the red tetrazoilium salt WST to a water solubleformazan dye was determined according to the manufacturer's instructions(Boehringer). Target cells (LNCaP and DU 145 as control) were seeded at2.5×10⁴/well of a 96-well plate and grown for 24 hours until a confluentcell layer was formed. Various dilutions of the recombinant immunotoxinsin aliquots of 50 μl/well were added and the plates were incubated for48 hours at 37° C., 5% CO₂. After this time the cultures were pulsedwith 15 μl/well WST reagent and incubated for 90 min at 37° C., 5% CO₂.Then the spectrophotometrical absorbances of the samples were measuredat 450 nm (reference 690 nm). The immunotoxin concentration required toachieve a 50% reduction in cell viability relative to that of untreatedcontrol cultures (50% inhibitory concentration=IC50) was calculated.

Cytotoxicity assays (WST) with the immunotoxins E8-P40 and A5-P40 wereprepared with PSMA expressing LNCaP cells and DU 145 control cells. Asshown in FIG. 11 a high cytotoxic effect could be shown with theimmunotoxin E8-PE40 on LNCaP cells with a IC50 value of 0.05 nM. In FIG.12 the cytotoxic effect of the immunotoxin A5-PE40 is shown with an IC50of about 0.09 nM. The cytotoxic background on not PSMA expressing DU 145cells was 5% for the E8 construct and only 0.01% for the A5 constructevidencing a very good therapeutic window.

EXAMPLE 13 Generation of the scFv H12 and D7 from mAb 3/F11 and 3/E7

From each mAb a scFv expression library in the phagemid pSEX wasgenerated as described in Example 5.

Production and selection of antibody-displaying phage was done accordingto Example 6.

After six panning rounds alternatively on PSMA and LNCaP cells onespecific positive clone was obtained, from mAB 3/E7, which was named H12and one positive clone was obtained form from mAB 3/F11, which was namedD7. The coding region of each scFv was transferred into the expressionvector pHOG-21.

ScFv expression and purification was done as described in Example 7.

EXAMPLE 14 Characterization of the scFv H12 and D7 a) Flow Cytometry onPSMA-Positive and -Negative Cell Lines

The scFvs H12 and D7 reacted with viable LNCaP cells as measured by flowcytometry.

From the saturation curves the antibody concentration reaching 50%saturation of PSMA sites was determined to be approximately 120 nM (H12)and 20 nM (D7) respectively. At saturating concentrations MFI values of70 (H12) and 40 (D7) were reached (FIG. 15).

To evaluate the PSMA binding specificity of the scFv H12 and D7,PSMA-negative prostate cancer cells of DU145 and PC3 and other PSMAnegative cell lines (HeLa, MCF7, HCT15 and Jurkat) were additionallystained and analyzed by flow cytometry. All three scFv did not stain thePSMA-negative cells.

b) Flow Cytometry on PSMA Transfectants

To verify a PSMA-specific binding, the scFv H12 and D7 were also testedon BOSC-23 cells transfected with PSMA. Both scFv showed a concentrationdependent binding to BOSC cells transfected with full-length PSMA butnot to non-transfected cells (FIG. 16). Saturating conditions werereached at 100 nM (D7) and 200 nM (H12). Similar to the mAbs, MFI-valueson the transfectants were lower than on LNCaP cells and showed a broaddistribution, which may correspond to varying PSMA molecules on the cellsurface of the former.

c) Immunofluorescence Cytology

Immunofluorescence cytology was prepared as described in Example 4.After detection with a laser scanning confocal microscope a strongbinding of the scFv to LNCaP cells and also an internalization intothese cells was observed.

d) ELISA and Western Blotting

Binding of the scFv H12 and D7 to purified PSMA in an solid phase ELISAand by Western blotting was weak.

The sequences (amino acid and nucleic acid) of H12 and D7 are given inFIG. 20 and FIG. 21.

TABLE 3 Characteristics of the anti-PSMA scFv H12 and D7 FACS on SEQ IDNO of FACS PSMA- SEQ ID NO of SEQ ID nucleic acid on transfected nucleicacid NO of sequence Original LNCaP BOSC Blot on sequence amino acid(complementary scFv mAb MFI* (MFI*) PSMA (coding strand) sequencestrand) H12 3/E7 70 25 100 kD 19 20 23 D7 3/F11 40 24 100 kD 21 22 24*MFI = Mean fluorescence intensity values at saturating conditions aftersubtraction of the background staining with an irrelevantisotype-matched control antibody or anti-mouse immunoglobulin alone.

EXAMPLE 15 Construction and Cytotoxicity of a H12-PE40 Immunotoxin andD7-PE40 Immunotoxin

Construction of the H12-PE40 and the D7-PE40 immunotoxin was similar toA5 and E8 immunotoxins described in example 11. PE-40 represents thePseudomonas exotoxic fragment.

Cytotoxicity was tested as described in example 12.

The immunotoxin promoted death of LNCaP cells in a time-dependentmanner; highest cytotoxic effects could be observed after 48 hincubation.

At this time IC₅₀ values of about 200 pM were found for H12-PE40 andD7-PE40 (FIG. 17).

Additionally, cytotoxicity of H12-PE40 and D7-PE40 was tested on thePSMA-negative cell lines DU 145, PC-3, MCF7 and HCT 15. No cytotoxicitywas found on these cell lines at concentrations up to 25 000 pM.

EXAMPLE 16 Construction of an Anti-PSMA/CD3 Diabody

A bispecific diabody specific for PSMA and the CD3 chain of the T cellreceptor complex was generated. The bispecific diabody was expressed inE. coli using a vector containing the dicistronic operon for cosecretionof VhCD3-VIA5 and VhA5-VICD3 scFv (FIG. 18). For the anti-A51CD3 diabodyconstruction the plasmids pKID19x3 and pKID 3x19 were used [Kipriyanov,Int. J. Cancer 1998, pp 763]. Bacterial periplasmatic expression andpurification was similar to the scFv.

EXAMPLE 17 Induction of Specific Cytotoxicity by Diabody A5-CD3

The ability of the bispecific diabody to induce tumor cell lysis byredirecting T cell-mediated cytotoxicity was investigated using PBMCfrom healthy donors as effector cells. After incubation with or withoutIL-2 for 4 days, the cells were added to LNCaP target cells, which wereseeded at 1.5×10⁴ cells/well of a 96-well plate. The effector-targetratio was 10:1. Diabody was added at different concentrations. Afterincubation of 48 hours the cultures were pulsed with 15 μl/well WSTreagent and incubated for 90 min at 37° C. and 5% CO₂. Then thespectrophotometrical absorbances of the samples were measured at 450 nm(reference 690 nm).

In this in vitro test the diabody appeared to be quite potent inretargeting activated and inactivated PBMC to lyse the target LNCaPcells in a concentration dependent manner (FIG. 19).

EXAMPLE 18 Diabody A5-A5

This bivalent monospecific diabody was generated similar to the A5-CD3diabody (example 16). Bacterial periplasmatic expression andpurification was similar to the scFv.

By flow cytometry a strong and specific binding of diabody A5-A5 toLNCaP cells could be shown.

1) An isolated monoclonal antibody or an antigen binding portion thereofwhich a) binds to prostate specific membrane antigen in its native formoccurring on the surface of tumor cells b) can be internalized by atumor cell, c) binds strongly to LNCAP cells but not or only minimallyto cells which lack expression of prostate specific membrane antigen andd) characterized in that it is linked to a label or a cytotoxic agent.2) Isolated monoclonal antibody or antigen binding portion thereofaccording to claim 1 characterized in that the PE fluorescence intensity(MFI) of the mAb is higher than 1000 and of the scFv higher than 40 atantigen saturation. 3) Isolated monoclonal antibody or an antigenbinding portion thereof according to claims 1 and 2 which show a highbinding activity to LNCAP cells reaching 50% saturation of PSMA sites atconcentrations between 1 nM and 120 nM. 4) Isolated monoclonal antibodyor an antigen binding portion thereof according to any of claims 1-3characterized in that the label is a particle which emits radioactive orfluorescence radiation. 5) Isolated monoclonal antibody or antigenbinding portion thereof according to claims 1-4 characterized in thatthe cytotoxic agent is a cell toxic substance selected from the groupconsisting of toxins, in particular taxol, cytocalasin B, gramicidin D,ethidium bromid, emetine, mitomycin, etopside, tenopside, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy antracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosteron,glycocorticoids, procain, tetracaine, lidokaine, propranolol and/orpuromycin. 6) Isolated monoclonal antibody or an antigen binding portionthereof according to any of claims 1 to 5 characterized in that itcomprises a partial amino acid sequence of at least 10 consecutive aminoacids of SEQ ID NO:1. 7) Isolated monoclonal antibody or antigen bindingportion thereof according to claim 6, characterized in that it comprisesat least one of SEQ ID NO:2-SEQ ID NO:7. 8) Isolated monoclonal antibodyor an antigen binding portion thereof according to any of claims 1 to 5characterized in that it contains a partial amino acid sequence of atleast 10 consecutive amino acids of SEQ ID NO:10. 9) Isolated monoclonalantibody or antigen binding portion thereof according to claim 8,characterized in that it comprises at least one of SEQ ID NO:11-SEQ IDNO:16. 10) Isolated monoclonal antibody or antigen binding portionthereof according to any of claims 1 to 5 characterized in that itcontains a partial amino acid sequence of at least 10 consecutive aminoacids of SEQ ID NO:20. 11) Isolated monoclonal antibody or antigenbinding portion thereof according to claim 10 characterized in that itcomprises a partial amino acid sequence of at least 25 consecutive aminoacids of SEQ ID NO:20. 12) Isolated monoclonal antibody or an antigenbinding portion thereof according to any of claims 1 to 5 characterizedin that it comprises a partial amino acid sequence of at least 10consecutive amino acids of SEQ ID NO:22. 13) Isolated monoclonalantibody or an antigen binding portion thereof according to claim 12characterized in that it comprises at least 25 consecutive amino acidsof SEQ ID NO:20. 14) Pharmaceutical composition comprising an isolatedmonoclonal antibody or an antigen binding portion thereof according toany of the preceding claims. 15) Use of an isolated monoclonal antibodyor an antigen binding portion thereof according to any of claims 1-13for the preparation of a medicament for the treatment of cancer. 16)Diagnostic kit for the detection of tumor cells comprising an isolatedmonoclonal antibody or an antigen binding portion thereof according toany of claims 1-13. 17) A method for the in vitro identification oftumor cells characterized in that the tumor cells to be identified arecontacted with an isolated monoclonal antibody or an antigen bindingportion thereof according to any of claims 1-13. 18) Use of an isolatedmonoclonal antibody or an antigen binding portion thereof according toany of claims 1 to 13 for the diagnostic identification of tumor cells.19) Isolated polynucleotide characterized in that it comprises acontiguous sequence of at least 20 nucleotides of any sequence of thegroup consisting of SEQ ID NOS: 8, 9, 17, 18, 19, 21, 23 and 24.